The goal of this second revised application is to investigate the ontogenetic mechanisms that underlie cholinergic synaptogenesis in the inner ear. Although synapse formation is best understood at the neuromuscular junction (NMJ), the applicability of this cholinergic model to other synapses in the nervous system has proved difficult. We propose to investigate cholinergic synapse formation, maturation and maintenance using vital, molecular and immunocytochemical methods in normal and transgenic animals as well as in organotypic culture. Specifically, we will explore the spatiotemporal expression, acquisition and loss of nicotinic receptors (formation), the localization of receptor clusters to cholinergic synapses (differentiation), and how these postsynaptic receptor clusters are maintained. Our basic hypothesis that bi-directional signaling between hair cells and efferent nerve terminals regulates the formation, differentiation and maintenance of cholinergic synapses, will be tested. Specific aim 1 investigates the hypothesis that the formation of nicotinic acetylcholine receptors (nAChRs) is independent of efferent innervation. Using embryonic mutant animals and organotypic culture explants from embryonic inner ears, we will characterize the expression and formation of nAChRs in the absence of cholinergic input. We will characterize the relation between the differential expression of nAChR subunits, the development of nAChR clusters, and efferent innervation. We will identify the time of appearance, cellular distribution, and degree of co-localization between nAChRs and presynaptic cholinergic terminals. We will also investigate the formation of nAChRs in vestibular hair cells. Specific aim 2 investigates the hypothesis that the differentiation of postsynaptic nicotinic receptors depends on cholinergic innervation. In postnatal animals, we will determine if nAChRs colocalize with medial or lateral olivocochlear neurons and the extent of colocalization between nAChR clusters and cholinergic innervation. We will investigate whether postsynaptic differentiation occurs after a loss of cholinergic input. Specific aim 3 investigates the hypothesis that the maintenance of postsynaptic nAChR clusters is dependent on medial efferent innervation. In mature and postnatal animals, we will characterize the loss of nAChR expression and clusters after elimination of cholinergic input. Specific aim 4 investigates the hypothesis that nAChR clustering in hair cells involves similar signaling molecules as found at the NMJ. We will investigate nAChR expression and clustering in transgenic mutant models that lack agrin, rapsyn or MuSK, all shown to be key factors in clustering at the NMJ. We will also characterize interactions between the hair cell nAChRs and these NMJ signaling molecules. In summary, these studies of cholinergic synapse development in the inner ear should have broad implications for the study of synapse formation throughout the nervous system as well as for a range of specific disorders associated defective cholinergic neurotransmission.